摘要：

目的： 探讨镁离子对大鼠骨髓间充质干细胞（bone marrow stromal cells，BMSCs）成骨分化的影响及其相关的机制。方法： 以成骨诱导培养液（含镁离子0.8 mmol/L）为对照组，在此培养液中依次添加100、200、400 μmol/L镁离子作为实验组（Mg 100、200、400 μmol/L组）。培养大鼠BMSCs，应用MTT法检测镁离子对细胞代谢活性的影响，碱性磷酸酶（alkaline phosphatase，ALP）染色和对-硝基苯磷酸盐法检测细胞ALP蛋白的表达，茜素红染色和半定量分析检测细胞外基质钙盐的沉积情况，实时荧光定量分析检测ALP、骨钙素（osteocalcin，OCN）、骨桥素（osteopontin，OPN）、BMP-2及镁转运蛋白1（MagT1）基因的相对表达量，激光共聚焦观察OCN、LC3蛋白的表达水平，Western Blot检测OCN蛋白、自噬相关蛋白的表达水平，自噬双标腺病毒转染观察细胞自噬流情况，荧光素酶法检测细胞内ATP浓度。结果： 1、4、7 d时，实验组Mg 100、200、400 μmol/L对大鼠BMSCs的增殖活性无显著影响（P>0.05）；实验组Mg 100、200、400 μmol/L ALP染色和茜素红染色较对照组深染，ALP蛋白分泌和ARS基质矿化均较对照组增加，其中Mg 100 μmol/L组较对照组有显著性差异（P<0.01）；7 d时，实验组Mg 100、200、400 μmol/L的ALP、OCN、OPN、BMP-2和MagT1基因相对表达均较对照组上调，其中Mg 200 μmol/L组表达量最高（P<0.01）；4 d时，实验组Mg 100、200、400 μmol/L OCN蛋白免疫荧光染色较对照组均有一定程度增强，其中Mg 200 μmol/L组OCN蛋白荧光染色最强；Mg 200 μmol/L组LC3蛋白荧光染色增强，呈斑点状分布；4 d时，实验组Mg 100、200、400 μmol/L OCN和LC3 Ⅰ/Ⅱ蛋白表达提高，Mg 200 μmol/L组的蛋白条带染色最深、灰度值比最高。1 d时，AdPlus-mCherry-GFP-LC3B转染后Mg 200 μmol/L组双荧光共表达成斑点状分布；3、6 d时，实验组Mg 100、200、400 μmol/L细胞内ATP浓度较对照组均下降，且差异具有统计学意义（P<0.01），其中Mg 400 μmol/L组的细胞内ATP水平最低。结论： 生理范围内镁离子浓度增加对BMSCs增殖活性无显著影响，可以提高BMSCs成骨分化、矿化性能，其作用可能与MagT1的表达上调、细胞内ATP水平下降和自噬激活相关。

关键词: 镁离子, 骨髓间充质干细胞, 成骨分化, 自噬, 镁转运蛋白1

Abstract:

Objective: To investigate the effect of magnesium ion on osteogenic differentiation of rat bone marrow stromal cells (BMSCs) and its mechanism. Methods: BMSCs cultured in the osteogenic medium （magnesium ion 0.8 mmol/L）was set as the control group. BMSCs cultured in the osteogenic medium with additional magnesium ion of 100, 200 and 400 μmol/L were set as experimental groups. The metabolic activity of BMSCs was detected by MTT, the expression of ALP protein was analyzed by alkaline phosphatase(ALP) staining and semi quantitative analysis. Alizarin red staining and semi quantitative analysis were used to detect extracellular matrix calcium deposition. The relative expression of ALP, OCN, OPN, BMP-2 and MagT1 gene was detected by real-time fluorescence quantitative analysis. The expression of OCN and LC3 protein was observed by laser confocal microscopy. Western Blot was used to detect the levels of OCN protein and autophagy related proteins. The autophagy flux was detected by fluorescence double labeling adenovirus transfection. The intracellular ATP level was detected by luciferase assay. Results: 1，4，7 d, Mg 100 μmol/L group, Mg 200 μmol/L group and Mg 400 μmol/L group had no significant effect on the proliferation of BMSCs（P>0.05）. ALP staining and ARS staining were stronger in Mg 100 μmol/L group, Mg 200 μmol/L group and 400 μmol/L group respectively. ALP and ARS activity quantitative assay demonstrated higher in Mg 100 μmol/L group, 200 μmol/L group and 400 μmol/L group, especially in Mg 100 μmol/L group（P<0.01）. 7 d, Mg 100 μmol/L group, 200 μmol/L group and 400 μmol/L group promoted the expression of ALP, OCN, OPN, BMP-2 and MagT1 genes, especially Mg 200 μmol/L group（P<0.01）. 4 d, the immunofluorescence staining of OCN protein in 100 μmol/L group, 200 μmol/L group and 400 μmol/L group was stronger than that in the control group, among which the OCN protein fluorescence staining was the strongest in Mg 200 μmol/L group. LC3 protein fluorescence staining in Mg 200 μmol/L group was enhanced and the dot distribution was formed. 4 d, Mg 100 μmol/L group, 200 μmol/L group and 400 μmol/L group enhanced the expression of OCN protein and the level of autophagy related proteins. 1 d, after AdPlus-mCherry-GFP-LC3B transfection, fluorescence co-expression dot was higher in Mg 200 μmol/L than that in the control group. 3 d, 6 d, Mg 100 μmol/L group, 200 μmol/L group and 400 μmol/L group decreased the concentration of intracellular ATP level （P<0.01）, especially Mg 400 μmol/L group. Conclusion: In physiological range, the increase of magnesium ion concentration had no significant effect on the proliferation of BMSCs, but promotes the osteogenic differentiation and mineralization of BMSCs. The mechanism may be related to the increase of MagT1 expression, the decrease of intracellular ATP concentration and activation of autophagy.

Key words: magnesium ion, bone marrow stromal cells, osteogenic differentiation, autophagy, MagT1